Crystal Structure of Rice α-Galactosidase Complexed with D-Galactose

Abstract

␣-Galactosidases catalyze the hydrolysis of ␣-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice ␣-galactosidase has been determined at 1.5Å resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of ␣-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (␤/␣) 8 -barrel structure, and the C-terminal domain was made up of eight ␤-strands containing a Greek key motif. The structure was solved as a complex with D-galactose, providing a mode of substrate binding in detail. The D-galactose molecule was found bound in the active site pocket on the C-terminal side of the central ␤-barrel of the catalytic domain. The D-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice ␣-galactosidase with chicken ␣-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes. ␣-Galactosidases (␣-Gals1 ; E.C. 3.2.1.22) catalyze the hydrolysis of ␣-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. ␣-Gals are widely distributed in animals, plants, and microorganisms.In humans, ␣-Gal is a lysosomal exoglycosidase that cleaves the terminal ␣-galactose residue from glycolipids and glycoproteins. Mutations in the ␣-Gal gene cause incomplete degradation of carbohydrates, resulting in Fabry disease (1, 2). In plants, galactomannan is one of the major storage polysaccharides in seeds, and ␣-Gal is one of the key enzymes in the degradation of cell wall galactomannan during germination (3, 4). Raffinose and stachyose in beans are known to cause flatulence, and ␣-Gal has the potential to alleviate these symptoms (5). In the sugar beet industry, ␣-Gal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (6).We have purified and sequenced several ␣-Gals from Mortierella vinacea, Penicillium purpurogenum, Thermus sp. T2, and Oryza sativa L. and have elucidated the substrate specificities of these enzymes using two types of the galactomannooligosaccharides, 63 -mono-␣-D-galactopyranosyl-␤-1,4-mannotriose and 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotetraose (7-13). The results showed that ␣-Gals have a diverse preference for substrates. The M. vinacea ␣-Gal I and yeast ␣-Gals were specific only for 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotriose, which has an ␣-galactosyl residue (designated the terminal ␣-galactosyl residue) linked to the non-reducing end mannose of ␤-1,4-mannotriose (14, 15). Aspergillis niger ␣-Gal and P. purpurogenum ␣-Gal, however, showed a preference for 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotetraose, which has an ␣-galactosyl residue (designated the side-chain ␣-galactosyl residue) attached to the inner mannose of ␤-1,4-ma...

Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves

“…The active site of family 27 acid a-Gals was predicted to contain two carboxyl groups (Mathew and Balasubramaniam, 1987), one serving as the catalytic nucleophile and the other one as the general acid/base catalyst. Recently, crystallization of a rice a-Gal confirmed the identity of Asp in the conserved motif LKYDNC after the fourth b-sheet as the catalytic nucleophile as shown previously for green coffee bean a-Gal (Ly et al, 2000;Fujimoto et al, 2002Fujimoto et al, , 2003. Further, Asp in the motif DIXD was shown to be the acid/base catalyst, and Trp in the conserved motif TPPMGWNSWN is discussed to function in hydrophobic substrate binding of the glycosyl oligosaccharids (Fujimoto et al, 2003).…”

“…Recently, crystallization of a rice a-Gal confirmed the identity of Asp in the conserved motif LKYDNC after the fourth b-sheet as the catalytic nucleophile as shown previously for green coffee bean a-Gal (Ly et al, 2000;Fujimoto et al, 2002Fujimoto et al, , 2003. Further, Asp in the motif DIXD was shown to be the acid/base catalyst, and Trp in the conserved motif TPPMGWNSWN is discussed to function in hydrophobic substrate binding of the glycosyl oligosaccharids (Fujimoto et al, 2003). When GGT was modeled in the known structure of rice a-Gal (SWISS-MODEL; http://swissmodel.expasy.org/), Asp-162 and Asp-217 as well as Trp-45 of GGT precisely superimposed over the respective Asp and Trp residues of rice a-Gal mentioned above (Fig.…”

“…This classification not only reflects substrate specificities and molecular mechanisms but also structural features of these enzymes. Hydrophobic cluster analysis of the GGT peptide sequence revealed that the catalytic domain of GGT comprised of an (a/b) 8 -barrel and a C-terminal domain made up of eight b-strands containing a Greek key motif, just like the plant acid a-Gal from rice (Fujimoto et al, 2003). The active site of family 27 acid a-Gals was predicted to contain two carboxyl groups (Mathew and Balasubramaniam, 1987), one serving as the catalytic nucleophile and the other one as the general acid/base catalyst.…”

A β-l-Arabinopyranosidase from Streptomyces avermitilis Is a Novel Member of Glycoside Hydrolase Family 27

“…Homology models of the first two domains and the C-terminal carbohydrate-binding module family 13 (CBM13) of SaArap27A were built with the MODELER program (Accelrys Software Inc., San Diego, CA) using the known crystal structures of rice ␣-galactosidase and CBM13 of Streptomyces olivaceoviridis ␤-xylanase (SoCBM13), respectively, as reference models (25,26). The first run of the MOLREP program (27) in the CCP4 program suite (28) using the ␣-galactosidase-derived model as the reference against the native data resulted in two prominent solutions yielding an R factor of 0.553.…”

“…Enzymatic Properties-␤-L-Arabinopyranosidase activity was determined using a mixture containing 25 . One unit of enzyme activity is defined as the amount of enzyme that released 1 mol of PNP per minute.…”